The human polyomavirus JC virus (JCV) causes the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML) in immunocompromised individuals, especially patients with Acquired Immunodeficiency Syndrome (AIDS). JCV infects tissues of the kidney, and is persistently shed during the lifetime of the individual. Up to 39% of the population is seroreactive to JCV, and viral DNA can be detected in up to 50% of normal kidney tissue. While infection in immunocompetent individuals is generally thought to be asymptomatic, reactivation of JCV in immunocompromised individuals results in dissemination of JCV to the brain and lytic infection of oligodendrocytes, resulting in PML. PML is present in about 5% of AIDS patients and before the introduction of anti-retroviral therapy, the prognosis was usually fatal within 6 months. With the advent of effective antiretroviral therapy, mortality rates have fallen, but PML is still fatal in about 50% of patients. More recently, immunomodulatory treatments that manage multiple sclerosis have unexpectedly resulted in PML. Currently, few options exist for the treatment of PML as a result of a poor understanding of viral pathogenicity of JCV. This project proposes to increase our understanding of JCV pathogenesis, by studying exposure and delivery of the viral genome to the host cell nucleus. Since JCV must balance between protecting its viral genome and allowing the host replication machinery to access its encapsidated genome for viral replication, the factors that regulate viral genome release are critical for successful infection. The first aim of this proposal will examine the cellular location of JCV uncoating by generating JCV virions whose capsid and genome are labeled with spectrally distinct fluorophores. Trafficking of these particles during infection will be performed in relation to fluorescently labeled cell markers by live cell confocal fluorescence microscopy. The second aim will investigate the nuclear import of JCV through the nuclear pore complex (NPC), specifically characterizing how these particles, whose diameter is greater than the upper limit of the NPC, are able enter the nucleus. The third aim proposes to reduce the diameter of a viral pore in the capsid and determine the effects on viral infectivity. This aim may provide a mechanistic understanding of release of minor capsid proteins and genome are from JCV and may lead to the development of antiviral compound that prevent the egress of the minor capsid proteins or genome through this pore. Taken together, these aims will provide critical information on viral pathogenesis of JCV and will identify new targets for the development of novel anti- viral therapies.